Polymicrobial infections have been linked to important human diseases including chronic wound infections, lung infections, and bacteremia, and can involve different species of bacteria, or bacteria and viruses, or bacteria and eukaryotes. Although the existence of co-pathogens in certain types of disease is established, the complexity of studying co-pathogen interactions using models of pathogenesis has limited our understanding of the role of the different pathogens in these diseases. The scientific premise of the proposed studies is that although polymicrobial infections are linked to infectious diseases, prior experimental studies have focused on single pathogens and have typically not considered the host:pathogen interactions in the presence of co- pathogens or with other members of the host's microbiota. Thus, we will consider not only the impact of co- pathogens on diarrheal illness and lung infections, but will also expand traditional laboratory assays, as well as tissue culture and mouse models to more closely recapitulate the complexity of host:pathogen interactions. We will investigate Aeromonas, E. coli, and/or Shigella in diarrheal co-infections, and the contributions of influenza virus and Streptococcus pneumoniae to respiratory co-infections. In Aim 1 we will use comparative genomics and metagenomics to provide insight into whether there are genes/genotypes, or microbial communities associated with diarrhea and/or co-pathogen interactions of these diarrheal pathogens. In Aim 2 we will further investigate the significance of diarrheal co-pathogens using dual transcriptomics on both the host and bacterial sides to study the impact of co-pathogen interactions in human intestinal enteroids and a mouse model with a simplified intestinal microbiota. In Aim 3, we will diversify our studies of co-infections by characterizing the impact of influenza virus on S. pneumoniae infection of the respiratory tract by performing dual transcriptional analyses with an ex vivo human lung epithelial cell model and an in vivo mouse model of lung infection. Our central hypothesis is that the interaction of multiple pathogens, bacterial-bacterial or bacterial-viral, can result in altered patterns of virulence of each of the pathogens, which will in turn influence interactions with the host, and have a significant role in the disease outcome. We further hypothesize that host responses differ between mono-infections and co-infections. We anticipate that upon completion of the studies we will have identified: (i) differences in the transcriptional profiles of the pathogens and the host in co-infection compared with mono- infection, (ii) genes that were not previously known to be involved in pathogenesis for these important pathogens, and (iii) novel host pathways driving immune and other responses to co-infections. Overall, the findings of this study will advance knowledge of the role of bacterial-bacterial and bacterial-viral co-pathogens in lung infections and diarrheal illness, and will enable future studies of the role of specific eukaryotic and prokaryotic genes and pathways in co-pathogen and/or host:pathogen interactions involved in these diseases. These studies will identify targets for future investigation as novel therapeutic interventions.